Tetraploidy is Associated with Poor Prognosis at Diagnosis in Multiple Myeloma

Cytogenetic assessment of plasma cells (PCs) provides prognostic information for patients with multiple myeloma (MM) and is most commonly carried out using fluorescence in-situ hybridization (FISH) testing or conventional karyotype analysis, although the latter is currently less commonly used.^1–3 Hyperdiploidy, with trisomies of odd numbered chromosomes typically portends a favorable prognosis, while hypodiploidy, certain translocations of immunoglobulin heavy chain (IgH) locus [t(4;14), t(14;16) and t(14;20)], deletion of 17p/monosomy 17 and gain of 1q are adverse prognostic factors.^1–11 Tetraploidy, which refers to a doubling of the diploid genome, is less commonly observed in MM.^5,12,13 There is limited data on the frequency of this finding and its prognostic impact in MM.¹⁴ In this report, we describe the incidence of tetraploidy in patients with newly diagnosed MM using a flow cytometry based method described below. We also report on genetic abnormalities that are associated with tetraploidy, as well as survival outcomes in this group compared to patients without tetraploidy.

We evaluated 275 consecutive patients with symptomatic MM diagnosed from 4/2/2012 to 12/03/2015 who underwent PCPRO testing before start of first-line therapy and they comprise the study population. We have developed a method of assessing ploidy of plasma cells, the Plasma Cell Proliferative Index (PCPRO), which is a flow cytometry based method that can estimate the DNA index of PCs. [Sidana et al. Manuscript in press Dec 2018] This is done by calculating the ratio of DNA content of clonal PCs to polytypic PCs, which serve as a control in each patient. PCPRO can also measure percentage of plasma cells in S-phase of cell cycle¹⁵, thereby providing an estimate of proliferative rate. The detailed procedural method to determine DNA index and S-phase using PCPRO is described in the supplementary material. DNA index values of 0.95–1.05 were considered diploid. Values below 0.95 were considered hypodiploid and values between 1.06 – 1.50 were considered hyperdiploid. Values from between 1.51 – 1.7 were considered near-tetraploid and values >1.7 with >10% clonal G2M cells and a visible 4n population were considered tetraploid. Conventional karyotype analysis and plasma cell FISH results are also reported. FISH testing was carried out by interphase cytoplasmic immunoglobulin FISH (cIg-FISH) for plasma cell disorders as previously described.^8,16

Statistical analysis was carried out using the JMP (version 13, SAS Institute Inc., Cary, NC). Overall survival (OS) and progression free survival (PFS) were estimated by the Kaplan Meier method and log-rank test was used to compare survival curves to evaluate prognostic impact of tetraploidy on survival. Chi-Square and Fischer Exact tests were used to carry out univariate analysis for categorical variables and Wilcoxon Rank Sum/Kruskal Wallis for continuous variables.

Of the 275 patients with NDMM, tetraploidy by PCPRO was observed in 17 (6%) patients and the remaining 258 (94%) patients were non-tetraploid (hyperdiploid: n=147, 53%; diploid: n= 98, 36%; hypodiploid: n=10, 4%, near-tetraploid: n=3, 1%). In the tetraploid group, a tetraploid clone or sub-clone was observed in 71% (12/17) patients by concurrent FISH testing. Conventional karyotyping revealed a normal karyotype in 71% (12/17) patients and complex karyotype in 24% (4/17) of patients and deletion 20q in the remaining one patient. (Supplementary Table S1)

Table 1 describes the baseline characteristics, as well as treatment and response to therapy in patients with tetraploidy vs. non-tetraploidy by PCPRO. There was no difference in the age at diagnosis and the proportion of males versus females between the two groups. There was no statistically significant difference in the ISS stage distribution in the two groups, although fewer patients with tetraploidy were observed to have ISS stage 1 disease (13% vs 30%). Patients in the tetraploidy group were observed to have higher median bone marrow plasma cells compared with the non-tetraploidy group (70% vs. 40%, p=0.002). They were also more likely to have high-risk genetic abnormalities [defined as deletion 17p/monosomy 17, t(4;14), t(14;16) and t(14;20)] on concurrent FISH testing (59% vs. 19%, p<0.001). In particular, patients with tetraploidy had a significantly higher incidence of deletion 17p/monosomy 17 (29% vs. 11%, p=0.048) and t(14;16) (24% vs. 2%, p<0.001). Patients with tetraploidy also had higher rates of t(11;14) (47% vs. 22%, p=0.03) and 1q gain (24% vs. 8%, p=0.05) compared with non-tetraploid patients. Monosomy 13 was also more common in the tetraploid group (71% vs. 35%, p=0.003), though no significant difference was observed for presence of 13q deletion (6% in both, p>0.99). On the other hand, tetraploid patients were less likely to harbor trisomies of odd numbered chromosomes (12% vs. 64%, p<0.001). The median percentage of plasma cells in S-phase was numerically higher in the tetraploidy group, indicating a slightly higher proliferative rate, but it did not meet statistical significance (1.2% vs. 0.9%, p=0.06).

Table 1:

Baseline Characteristics and Treatment

	Tetraploid, N=17 n/N (%), or median (IQR)	Non-tetraploid, N=258 n/N (%) or median (IQR)	P value
Age, years	72 (62–80)	67 (60–74)	0.2
Males	13/17 (76)	157/258 (61)	0.2
ISS stage I/II/III	2/7/7; N=16 (13/44/44)	70/94//69; N=233 (30/40/30)	0.2
BMPC percentage	70 (50–85)	40 (20–66)	0.002
S-phase	1.2 (0.8–2.1)	0.9 (0.5–1.6)	0.06
S-phase ≥ 2%	4/17 (24)	50/254 (20)	0.8
FISH findings
High risk cytogenetics	10/17 (59)	49/248 (19)	<0.001
Deletion 17p/monosomy 17	5/17 (29)	27/246 (11)	0.048
t(4;14),	1/17 (6)	23/248 (9)	>0.99
t(14;16)	4/17 (24)	5/248 (2)	0.001
t(14;20)	1/17 (6)	1/248 (0.5)	0.1
Other cytogenetic abnormalities
t(11;14)	8/17 (47)	53/245 (22)	0.03
Gain1q	4/17 (24)	19/246 (8)	0.05
Trisomies	2/17 (12)	155/244 (64)	<0.001
Monsomy 13	12/17 (71)	86/246 (35)	0.003
Deletion 13q	1/17 (6)	15/246 (6)	>0.99
Treatment and Response
Proteasome inhibitor in first-line therapy	14/17 (82)	182/258 (71)	0.3
ASCT as part of first-line therapy	6/17 (35)	102/258 (40)	0.7
Hematologic response: VGPR or better	12/16 (75)	143/209 (68)	0.6

Abbreviations: ASCT: Autologous stem cell transplant; BMPC: Bone marrow plasma cells; FISH: fluorescence in-situ hybridization; ISS: International Staging System; VGPR: Very good partial response

P values ≤0.05 are in bold. High risk cytogenetics is defined by presence of deletion 17p/monosomy 17, t(4;14), t(14;16) and t(14;20)

All treated patients received novel agent based treatment with a proteasome inhibitor based regimen being used in 82% vs. 71% (p=0.3) patients in the tetraploidy vs. non-tetraploidy groups. Similar rates of upfront transplant were observed across the two groups (35% vs. 40%, p=0.7). No difference was observed in the rates of best hematologic response to first-line therapy, with rates of very good partial response or better in the tetraploid vs. non-tetraploid group being 75% vs. 68%, respectively (p=0.6).

Median follow-up for all patients was 49 months from start of first-line therapy. The median OS in patients with tetraploidy at diagnosis vs. those without tetraploidy was 29 months vs. not reached, p<0.001, (Figure 1a). One and two year estimated OS in the tetraploidy group was 82% and 53% compared with 91% and 80% in the non-tetraploidy group, respectively. As patients with tetraploidy were more likely to have high-risk FISH abnormalities, a sub-group analysis was conducted for patients with high-risk FISH abnormalities. Even in the subset of patients who had high-risk FISH [defined as deletion 17p, t(4;14), t(14;16) and t(14;20)], patients with tetraploidy (n=10) had inferior OS compared to those without tetraploidy (n=49), with median OS of 25 vs. 54 months, p=0.04, respectively. (Figure 1b). In the standard risk group, patients with tetraploidy had numerically lower OS (40 months vs. not reached), but it did not quite reach statistical significance (p=0.09).

Figure 1: Survival analysis in newly diagnosed myeloma based on tetraploidy by PCPRO.

Figure 1a: All newly diagnosed multiple myeloma patients

Tetraploidy present, N=17, median OS: 29 months

Non-tetraploid, N=258, median OS: Not reached

P<0.001

Figure 1b: Patients with high risk FISH

Tetraploid, N=10, median OS: 25 months

Non-tetraploid, N=49, median OS: 54 months

P=0.04

In conclusion, tetraploidy resulting from a doubling of the diploid genome (4n) is observed in a small proportion of patients with MM, with 6% patients in our study demonstrating tetraploidy using a flow cytometry based method to assess ploidy. Patients with tetraploidy were more likely to have associated high-risk genetic abnormalities. Interestingly, simultaneous tetraploidy was noted in 12/17 patients on FISH testing. This discrepancy may be due to assessment of a limited number of cells for FISH evaluation compared to flow cytometry. Tetraploidy is often a subclonal finding on flow cytometry. Moreover, at our institution, FISH reporting requires that an abnormality be present in at unless 10% of cells. At present, there is limited information on prognostic implications of tetraploidy in myeloma by FISH testing or other techniques. We observed that tetraploidy is associated with a poor prognosis in patients with NDMM, even in the sub-group with high-risk FISH abnormalities. This finding is of significant interest as it may identify a sub-group of NDMM patients with poor prognosis, who may be candidates for more aggressive risk adapted therapy in future. However, as our sample size is small, this finding requires further validation in a larger cohort of patients.